In order for T cells to respond to foreign proteins, two signals must be provided by antigen-presenting cells (APCs) to resting T lymphocytes (Jenkins, M. and Schwartz, R. (1987) J. Exp. Med. 165, 302-319; Mueller, D. L., et al. (1990) J. Immunol. 144, 3701-3709). The first signal, which confers specificity to the immune response, is transduced via the T cell receptor (TCR) following recognition of foreign antigenic peptide presented in the context of the major histocompatibility complex (MHC). The second signal, termed costimulation, induces T cells to proliferate and become functional (Lenschow et al. 1996. Annu. Rev. Immunol. 14:233). Costimulation is neither antigen-specific, nor MHC restricted and is thought to be provided by one or more distinct cell surface molecules expressed by APCs (Jenkins, M. K., et al. 1988 J. Immunol. 140, 3324-3330; Linsley, P. S., et al. 1991 J. Exp. Med. 173, 721-730; Gimmi, C. D., et al., 1991 Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Young, J. W., et al. 1992 J. Clin. Invest. 90, 229-237; Koulova, L., et al. 1991 J.Exp. Med. 173, 759-762; Reiser, H., et al. 1992 Proc. Natl. Acad. Sci. USA. 89, 271-275; van-Seventer, G. A., et al. (1990) J. Immunol. 144, 4579-4586; LaSalle, J. M., et al., 1991 J. Immunol. 147, 774-80; Dustin, M. I., et al., 1989 J. Exp. Med. 169, 503; Armitage, R. J., et al. 1992 Nature 357, 80-82; Liu, Y., et al. 1992 J.Exp. Med. 175, 437-445).
The CD80 (B7-1) and CD86 (B7-2) proteins, expressed on APCs, are critical costimulatory molecules (Freeman et al. 1991. J. Exp. Med. 174:625; Freeman et al. 1989 J. Immunol. 143:2714; Azuma et al. 1993 Nature 366:76; Freeman et al. 1993. Science 262:909). B7-2 appears to play a predominant role during primary immune responses, while B7-1, which is upregulated later in the course of an immune response, may be important in prolonging primary T cell responses or costimulating secondary T cell responses (Bluestone. 1995. Immunity. 2:555).
One ligand to which B7-1 and B7-2 bind, CD28, is constitutively expressed on resting T cells and increases in expression after activation. After signaling through the T cell receptor, ligation of CD28 and transduction of a costimulatory signal induces T cells to proliferate and secrete IL-2 (Linsley, P. S., et al. 1991 J. Exp. Med. 173, 721-730; Gimmi, C. D., et al. 1991 Proc. Natl. Acad. Sci. USA. 88, 6575-6579; June, C. H., et al. 1990 Immunol. Today. 11, 211-6; Harding, F. A., et al. 1992 Nature. 356, 607-609). A second ligand, termed CTLA4 (CD152) is homologous to CD28 but is not expressed on resting T cells and appears following T cell activation (Brunet, J. F., et al., 1987 Nature 328, 267-270). CTLA4 appears to be critical in negative regulation of T cell responses (Waterhouse et al. 1995. Science 270:985). Blockade of CTLA4 has been found to remove inhibitory signals, while aggregation of CTLA4 has been found to provide inhibitory signals that downregulate T cell responses (Allison and Krummel. 1995. Science 270:932). The B7 molecules have a higher affinity for CTLA4 than for CD28 (Linsley, P. S., et al., 1991 J. Exp. Med. 174, 561-569) and B7-1 and B7-2 have been found to bind to distinct regions of the CTLA4 molecule and have different kinetics of binding to CTLA4 (Linsley et al. 1994. Immunity. 1:793). A new molecule related to CD28 and CTLA4, ICOS, has been identified (Hutloff et al. 1999. Nature. 397:263; WO 98/38216), as has its ligand, which is a new B7 family member (Aicher A. et al. (2000) J. Immunol. 164:4689-96; Mages H. W. et al. (2000) Eur. J. Immunol. 30:1040-7; Brodie D. et al. (2000) Curr. Biol. 10:333-6; Ling V. et al. (2000) J. Immunol. 164:1653-7; Yoshinaga S. K. et al. (1999) Nature 402:827-32). If T cells are only stimulated through the T cell receptor, without receiving an additional costimulatory signal, they become nonresponsive, anergic, or die, resulting in downmodulation of the immune response.
The mechanisms by which the immunoprivileged nature of the embryo is established and maintained during pregnancy are not fully understood; the role of embryonic costimulatory molecules in this process has not been addressed. In a murine model, maternal tolerance of embryonic antigens has been shown to be temporal and resensitization to these antigens are quickly reestablished after pregnancy (Bonney and Matzinger, 1997, J Immunol 158, 40-7). Despite this apparent transient tolerance, evidence indicates that dynamic immunological interactions occur at the embryonic/material interface between maternal NK cells and macrophages and embryonic tissues (Duclos et al., 1995; Am J Reprod Immunol 33, 354-66; Duclos et al., 1996, Biol Reprod 54, 1088-95; Duclos et al., 1994, Cell Immunol 159, 184-93).
Macrophages and NK cells are the predominant types of immune cells within the maternal decidua during early embryonic development while T cells accumulate during late development. In a recent report, cultured human decidual cells were found to be capable of antigen presentation (Olivares et al., 1997, Biol Reprod 57, 609-15). In an animal model of immune mediated spontaneous abortion, macrophage infiltration of the decidua is an early indication of immunological rejection and embryo resorption (Duclos et al., 1995, Am J Reprod Immunol 33, 354-66). It is generally thought that maternal/fetal cellular transfer is rare due to the chorionic cell layers that separate the two systems (Billington, 1992, Baillieres Clin Obstet Gynaecol 6, 417-38). However, evidence exists demonstrating that cross trafficking of cells between maternal and fetal systems occurs at very high frequency, albeit at very low levels. In a recent study in which mouse embryos were implanted into transgenic LacZ female mice, the presence of maternal LacZ positive cells in the embryo proper was reported (Piotrowski and Croy, 1996, Biol Reprod 54, 1103-10). In a separate transplantation study, it was found that hypoimmunity of recipient mice toward MHC haplotype mismatched maternal skin grafts was correlated with the low level presence of maternal T cells in the neonate, presumably acquired in utero, present in the lymph nodes of the recipient mice (Zhang and Miller, 1993, Transplantation 56, 918-21). These observations suggest that low level cellular infiltration of the embryo by maternal lymphocytes is possible during the course of the pregnancy.
Spontaneous early embryo resorption following implantation occurs in many species, but little is known regarding the causes or the prevention of early pregnancy failure. Further insight into the processes that result in spontaneous abortion and the development of improved methods for treating subjects who are at risk for spontaneous abortion would be of great benefit.
In one aspect, the invention provides a method of inhibiting spontaneous abortion in a subject by administering to the subject an agent that inhibits a costimulatory signal in a T cell such that spontaneous abortion in the subject is inhibited.
In one embodiment, the agent is selected from the group consisting of: an antibody to B7-1, an antibody to B7-2, an antibody to an ICOS ligand, and a combination of an antibody to B7-1 and an antibody to B7-2.
In another embodiment, the agent comprises a soluble form of CD28 or ICOS. In another embodiment, the agent comprises a soluble form of CTLA4. In one embodiment, the soluble form of CTLA4 is CTLA4Ig.
In one embodiment, the CTLA4Ig comprises a constant region domain that has been modified to reduce at least one effector-mediated function.
In one embodiment, the subject is a human.
In another embodiment, the subject is a domesticated animal.
In another embodiment, the subject is an endangered species.
In another embodiment, the subject is a non-human animal which is being used to carry cloned, non-human embryos.
In another aspect, the invention provides a method of down regulating an immune response by a subject to an embryo comprising: administering to a subject a therapeutically effective amount of a soluble CTLA4-Ig fusion protein such that an adverse immune response to the embryo is downregulate. In one embodiment, the subject is a human.
In one embodiment, the subject has had a previous spontaneous abortion.
In one embodiment, the soluble CTLA4-Ig fusion protein is administered to the subject prior to or at the time of implantation of the embryo.
In one embodiment, the method further includes administering to the subject soluble CTLA4-Ig fusion protein after implantation of the embryo.
In one embodiment, the CTLA4Ig comprises a constant region domain that has been modified to reduce at least one effector-mediated function.
In another aspect, the invention provides a method of enhancing the ability of a subject to carry at least one embryo to term comprising: administering to a subject a soluble CTLA4-Ig fusion protein such that the host immune response to the embryo is decreased.
In another embodiment, the invention provides a method of diagnosing a subject at risk for or suffering from immune-mediated spontaneous abortion which includes determining the level of one or more of an adhesion molecule (e.g., VCAM-1, P-selectin, and/or E-selectin), an inflammatory cytokine (e.g., IL-2, IL-10, IL-12, IL-11, TNFxcex1, IL-1xcex2, TGF, RANTES, IL-6, and/or WFN-xcex3), and/or an immune cell surface molecule (e.g., B7.1, B7.2, CD4, CD8, GL50, and/or ICOS). In another embodiment, the invention provides a method of determining whether treatment of a subject using the methods of the invention is having the desired effect, which includes determining the level of one ore more of an adhesion molecule, an inflammatory cytokine, and/or an immune cell surface molecule.